PROJECT SUMMARY Niemann-Pick type C (NPC) is a lysosomal storage disease that is inherited in an autosomal recessive manner. It is mostly caused by mutations in Npc1, a protein that export cholesterol out of the lysosomes, hence abnormal accumulation of cholesterol in lysosomes is a hallmark of NPC. Neurological symptoms of NPC include ataxia, cognitive impairment, and dementia that lead to premature death. Purkinje cells (PCs) are among the most susceptible neurons to Npc1 mutations leading to their early degeneration and development of early neurological symptoms such as clumsiness, gait defects and ataxia. The pathological changes in the cerebellum that precede and possibly precipitate Purkinje cells degeneration are not completely known. Studies in NPC animal models suggest that microglia reactivity and neuroinflammatory responses occur early during disease, but it is not completely clear if these pathological changes are caused by NPC1 deficiency or as a response to early PCs progressive degeneration. Given that neurological symptoms are accompanied by severe neurodegeneration, a major goal of the proposed study is to understand how NPC1 deficiency impacts the cerebellum during postnatal development, an asymptomatic stage in childhood NPC. A great portion of the anatomical and functional development of the cerebellum occurs postnatally. We are particularly interested in examined the postnatal development of the synaptic connections between PCs and climbing fibers (CF), which experience a long phase of pruning and refinement. Although the role of cortical microglia in developmental synaptic pruning is well recognized, their role in the synaptic refinement of cerebellar circuits is not known. Preliminary data in our laboratory indicate that NPC1 deficiency causes significant changes in microglial cells during postnatal developmental stages. Since the role of microglial cells and the effects of NPC1 deficiency in synapse formation and refinement have not been elucidated in the postnatal cerebellum, the goal of this proposal is to determine how NPC1 deficiency affects the phenotype of microglial cells as well as the synaptic structures and connections between PCs and presynaptic CFs. For the proposed study we have chosen the Npc1nmf164 mutant mouse, which presents a late-onset and slower disease progression than mice with complete deletion of Npc1. We will be investigating the following three aims: In Aim 1 we will investigate how Npc1nmf164 mutation alters microglia phenotype during postnatal stages of synaptic refinement and molecular pathways associated with early neuroinflammatory responses in the cerebellum will be identify. In Aim 2, we will specifically test whether microglia is involved in postnatal CF axon pruning. We will also investigate how Npc1nmf164 mutation affects the postnatal process of CFs pruning. In Aim 3, the effect of the Npc1nmf164 mutation in synapse formation and psychomotor behaviors will be established. Understanding the pathological events that precede PC degeneration and disease onset in NPC is critical for the designing and testing of effective therapeutic interventions that prevent or delay the progression of NPC.